Background: Chimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CART cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CART cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering. Methods: Two second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5 + malignant cell lines, primary CD5 + malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5 + T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo. Results: Both BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5 + malignant cells in vitro and prolonged the survival of TALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5 + malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK. Conclusions: Anti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cellassociated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.

BackgroundChimeric antigen receptor-engineered T (CAR-T) cells have extraordinary effect in treating lymphoblastic leukemia. However, treatment of acute myeloid leukemia (AML) using CAR-T cells remains limited to date. Leukemogenesis always relates with the abnormalities of cytogenetics, and nearly one third of AML patients have activating mutations in Fms-like tyrosine kinase 3 (FLT3) which reminded poor prognosis. Considering the FLT3 expressed in AML patients’ blast cells, it may be a new candidate target for CAR-T therapy to treat FLT3+ AML, especially patients harboring FLT3-ITD mutation.MethodsThe FLT3L CAR-T using FLT3 ligand as recognizing domain was constructed. The specific cytotoxicity against FLT3+ leukemia cell lines, primary AML cells, and normal hematopoietic progenitor stem cells (HPSCs) in vitro were evaluated. In addition, FLT3+ AML mouse model was used to assess the effect of FLT3L CAR-T therapy in vivo.ResultsFLT3L CAR-T cells could specifically kill FLT3+ leukemia cell lines and AML patients’ bone marrow mononuclear cells in vitro (with or without FLT3 mutation) and have more potent cytotoxicity to FLT3-ITD cells. In a human FLT3+ AML xenograft mouse model, FLT3L CAR-T cells could significantly prolong the survival of mice. Furthermore, it was found that FLT3L CAR-T cells could activate the FLT3/ERK signaling pathway of FLT3+ leukemia cells with wild-type FLT3; meanwhile, it had no inhibitory effects on the colony formation of CD34+ stem cells derived from normal human umbilical cord blood.ConclusionsThe ligand-based FLT3L CAR-T cells could be a promising strategy for FLT3+ AML treatment, especially those carried FLT3 mutation.Electronic supplementary materialThe online version of this article (10.1186/s13045-018-0603-7) contains supplementary material, which is available to authorized users.

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